N-Linked Glycosylation Is Required for Transferrin-InducedStabilization of Transferrin Receptor 2 but Not for Transferrin Bindingor Trafficking to the Cell Surface

摘要

Transferrin receptor 2 (TfR2) is a member of the transferrin receptor-like family of proteins. Mutations in TfR2 can lead to a rare form of the iron overload disease, hereditary hemochromatosis. TfR2 is proposed to sense body iron levels and increase the level of expression of the iron regulatory hormone, hepcidin. Human TfR2 (hTfR2) contains four potential Asn-linked (N-linked) glycosylation sites on its ectodomain. The importance of glycosylation in TfR2 function has not been elucidated. In this study, by employing site-directed mutagenesis to remove glycosylation sites of hTfR2 individually or in combination, we found that hTfR2 was glycosylated at Asn 240, 339, and 754, while the consensus sequence for N-linked glycosylation at Asn 540 was not utilized. Cell surface protein biotinylation and biotin-labeled Tf indicated that in the absence of N-linked oligosaccharides, hTfR2 still moved to the plasma membrane and bound its ligand, holo-Tf. However, without N-linked glycosylation, hTfR2 did not form the intersubunit disulfide bonds as efficiently as the wild type (WT). Moreover, the unglycosylatedform of hTfR2 could not be stabilized by holo-Tf. We further provideevidence that the unglycosylated hTfR2 behaved in manner differentfrom that of the WT in response to holo-Tf treatment. Thus, the putativeiron-sensing function of TfR2 could not be achieved in the absenceof N-linked oligosaccharides. On the basis of our analyses, we concludethat unlike TfR1, N-linked glycosylation is dispensable for the cellsurface expression and holo-Tf binding, but it is required for efficientintersubunit disulfide bond formation and holo-Tf-induced stabilizationof TfR2.